Abstract Congenital malformations, or structural birth defects, are now the leading cause of infant mortality in the US and Europe. Of the congenital malformations, congenital heart disease (CHD) is the most common. Understanding the molecular mechanisms of heart development is critical to develop intervention therapies for patients with CHD. Regulation of gene expression through transcriptional and epigenetic means is crucial during heart development, as gene misexpression leads to manifestations of CHD. Our lab has recently found that the cardiac-specific transcription factors TBX5 and TBX20, both causative to CHD, interact directly with the chromatin modifying Nucleosome Remodeling and Deacetylase (NuRD) complex through the ATP- dependent helicase subunit CHD4, potentially indicating a transcriptional regulatory role for the NuRD complex in the developing heart. While the composition and mechanism of the NuRD complex and CHD4 have been well-characterized in cell culture and limited tissue model systems, its requirement during mammalian heart development has yet to be established. Therefore, this proposal aims to determine the functional requirement of the cardiac NuRD complex in development by utilizing an in vivo approach. Preliminary data indicates that ablation of CHD4 in the developing heart is embryonically lethal at 12.5 days of gestation in a mouse model system, a stage equivalent to human mid-gestation. Phenotypic analysis using histological and ultrastructural methods indicates that CHD4 cardiac null hearts have myocardial development defects including alterations in cardiac sarcomere structure. Differential gene analysis via RNA sequencing of embryonic CHD4 null cardiac tissue revealed that ablating CHD4 from the developing heart results in misexpression of non-cardiac myofibril isoforms. The present study will (1) identify the cardiac cell type responsible for myocardial development defects, by generating a cardiomyocyte-specific deletion of CHD4 in the developing heart utilizing a cardiomyocyte specific Cre-recombinase driver. These CHD4 cardiomyocyte null embryos will be analyzed for myocardial development and cardiac function defects by histological methods and physiological assessments of cardiac function via embryonic echocardiography. This will identify the requirement for CHD4 in cardiomyocytes during myocardial development. This proposal will further (2) determine the requirement of CHD4 during embryonic heart development by identifying whether misexpression of skeletal and smooth muscle myofibril isoforms disrupt cardiac sarcomere formation and cardiomyocyte differentiation. This proposal will further identify whether these non-cardiac myofibril isoforms are direct targets of CHD4-mediated gene regulation in embryonic cardiac tissue by performing chromatin immunoprecipitation followed by high- throughput sequencing (ChIP-seq). Collectively, these studies will establish the role of CHD4 and the NuRD complex in cardiac development, determine the molecular mechanism of NuRD complex function in cardiomyocytes, and identify direct targets of CHD4 gene regulation in vivo.